The Her-2/neu or erbB-2 gene encodes a transmembrane protein that is a member of the type I family of growth factor receptors (Akiyama, T. et al., Science 232: 1644-1646, 1986). Amplification of this gene results in overexpression of the encoded 185 kDa receptor tyrosine kinase.
The Her-2/neu protein has been found to be amplified and overexpressed in several types of human adenocarcinomas, especially in tumors of the breast and the ovary. The overexpression was correlated with short relapse time and poor survival rate of breast cancer patients (Slamon, D. J. et al., Science 235: 177-182, 1987), suggesting that Her-2/neu overexpression likely plays a critical role in the development of human cancers. Several lines of evidence also support a direct role of Her-2/neu in the pathogenesis and clinical aggressiveness of Her-2/neu-expressing tumors (Kobayashi H. et al., Cancer Res. 60: 5228-5236, 2000). For example, Herceptin, a humanized anti-Her-2/neu monoclonal antibody used for treatment of Her-2/neu-expressing tumors, has been demonstrated to bring clinical benefits in advanced breast cancer patients (Ewer, M. S. et. al., Semin. Oncol. 26: 96, 1999). In addition, Her-2/neu-specific antibodies and T cells are detected in breast and ovarian cancer patients. Therefore, Her-2/neu oncogene is an excellent target for the development of therapeutic vaccines specific for Her-2/neu-overexpressing human cancers.
Since human Her-2/neu gene has tyrosine kinas activity in the intracellular domain and its overexpression itself stimulates abnormal cell division, there are several attempts to eliminate possible oncogenecity of Her-2/neu by introducing a mutation into the cytoplasmic kinase domain to inhibit tyrosine kinase activity or by constructing truncated Her-2/neu plasmids lacking the intracellular or extracellular domain (Wei, W. I. et al., Int. J. Cancer 81: 748-754, 1999)
Naked plasmids are attractive candidate vectors for the development of cancer vaccines encoding tumor-associated antigens. They are relatively simple to generate and safe to administer. Because they are not proteins nor associated with a viral coat, naked nucleic acids are not generally subject to neutralizing antibody reactions that can hamper the clinical efficacy of vaccines (Hellstrom, I. and Hellstrom, K. E., J. Immunother. 21: 119-126, 1998). In preclinical tumor models, DNA vaccines encoding rat (Chen, Y. et al., Cancer Res. 58: 1965-1971, 1998) or human Her-2/neu (Pilon, S. A. et al., J. Immunol. 167: 3201-3206, 2001) induced preventive efficacy against Her-2/neu expressing tumor cells.
Although successful preventive efficacy against Her-2/neu expressing tumor by DNA vaccination was achieved by many earlier experiments, no successful therapeutic efficacy was reported using only Her-2/neu expressing plasmids. The difficulty lies on the slow gain of antitumor immunity due to the lag time before antigenic expression of Her-2/neu expressing plasmids, while mammary tumor grows relatively fast. Therefore, some of the Her-2/neu therapeutic vaccine experiments were conducted based on the combination of DNA and cytokine-Secreting tumor cells (Chen, S. A. et al., Clin. Cancer Res. 6: 4381-4388, 2000), or dendritic cell (Chen, Y., Gene Ther. 8: 316-323, 2001).
Since a DNA vaccine has many advantages including mass-productivity, safety, and convenience (Gurunathan, S. et al., Annu. Rev. Immunol. 18:927-974, 2001), the present inventors have endeavored to develop Her-2/neu expressing plasmid constructs having high anti-cancer activity which can be effectively used as a DNA vaccine for preventing and treating cancer.